Optimization of protein removal process of Lonicera japonica polysaccharide and its immunomodulatory mechanism in cyclophosphamide‐induced mice by metabolomics and network pharmacology

Abstract In this study, TCA–n‐butanol was chosen as the best deproteinization method for Lonicera japonica polysaccharide (LJP) by comparing the polysaccharide retention rate and the protein clearance rate of five different methods. The response surface methodology (RSM) based on the Box–Behnken design (BBD) was used to optimize the deproteinization conditions as follows: TCA: n‐butanol = 1: 5.1, polysaccharide solution: (TCA–n‐butanol) = 1: 2.8, and shook for 33 min. LJP could promote the thymus and spleen indexes of cyclophosphamide (CTX)‐induced immune‐deficient mice. Besides, the contents of cytokine interleukin‐2 (IL‐2) and hemolysin in mice serum were augmented after treatment with LJP. Based on serum metabolomics analysis, a total of 14 metabolites (VIP >1.0, FC >2 or FC <0.5, and p value < .05) were selected as the potential biological biomarkers related to the LJP for treating CTX‐induced mice. After the pathway enrichment analysis, these metabolites were mainly involved in the relevant pathways of arginine biosynthesis, Citrate cycle, and other metabolic pathways. Network pharmacology further showed that there were 57 key targeting proteins in the intersection of the potential biological biomarkers and immunodeficiency‐related targeting proteins according to protein–protein interactions analysis (PPI). The biological function analysis indicated that the potential biological processes were mainly associated with tricarboxylic acid (TCA) cycle, phospholipid metabolic process, metabolic process, and so on. In conclusion, serum metabolomics combined with network pharmacology could be helpful to clarify the immunomodulatory mechanism of LJP and provide a literature basis for further clinical research on the therapeutic mechanism of LJP.


| INTRODUC TI ON
Lonicera japonica, also named "Jin Yin Hua" or "Ren Dong" in China, is a well-known traditional Chinese medicine (TCM) to treat fever, arthritis, respiratory infection, and epidemic disease (Liu, Fang, et al., 2018;Wang et al., 2017). Nowadays, some evidence indicate that Lonicera japonica might have a significant effect on COVID−19 patients by reducing fever and improving breathing (Zhao et al., 2021).
However, we found that polysaccharides extracted by water often contain more protein, which affects further study of their chemical properties and pharmacological activities (Cao et al., 2022;Younes & Rinaudo, 2015). To optimize the best approach and the best condition of deproteinization, five methods were investigated, including the enzymatic method, Sevage method, enzymatic-Sevage method, trichloroacetic acid (TCA) method, and TCA-n-butanol method.
Response surface methodology (RSM) was used to optimize protein removal conditions.
In recent years, the immunomodulatory activities of polysaccharides from TCM were one of the most critical biological activities. Although relevant literature reports show that Lonicera japonica polysaccharides had immunomodulatory functions, the mechanism of action is still not fully understood (Zhou et al., 2018).
Metabolomics is a new biological technology that provides a powerful method to study the biomarkers, the changes in biological systems, and is associated with physiological and pathophysiological processes. This method provides comprehensive qualitative and quantitative analysis of metabolites in vivo, providing vital information on chemical biomarkers, and could be used for determining taxonomy and quality control. Therefore, metabolomics could better demonstrate the key metabolites, pathways, and main regulatory processes of immunocompromised mice. Network pharmacology could explain the synergy of TCM with compounds and multiple target substances, thereby systematically revealing the pathogenesis and molecular understanding of drugs. Metabolomics combined with network pharmacology could clarify the mechanism and target of TCM at the level of systemic biology and whole integration (Ren et al., 2020).
In this study, Kunming mice were used to establish a CTX-induced immunodeficiency model and observe the immune enhancement effect of Lonicera japonica polysaccharides, and screen differential metabolites through serum metabolomics. In addition, we also combined network pharmacology to further explore the action mechanism of Lonicera japonica polysaccharides in immunomodulation. The process of the experiment is shown in Figure 1.  (Liu, Huang, & Hu, 2018;.

| Comparison of Lonicera japonica crude polysaccharide removal methods
When we investigated the effect of removing protein, the phenolsulfuric acid method and the Folin-phenol method were used to determine the content of polysaccharides and protein before and after protein removal (Cao et al., 2022). The polysaccharide retention rate, protein clearance rate, and comprehensive score were used as indicators to evaluate the effect of protein removal. The calculations were as follows: where m 1 is the polysaccharide weight of the Lonicera japonica crude polysaccharide after protein removal and m 2 is the polysaccharide weight of the Lonicera japonica crude polysaccharide before protein removal.
where m 3 is the protein weight of the Lonicera japonica crude polysaccharide after protein removal and m 4 is the protein weight of the Lonicera japonica crude polysaccharide before protein removal.
where W 1 is the polysaccharide retention rate, W 1,max is the maximum polysaccharide retention rate, W 2 is the protein clearance rate, and W 2,max is the maximum protein clearance rate.

| Enzymatic protein removal method
Four equal parts of crude polysaccharide samples were taken and dissolved in distilled water, then added 0.4% trypsin, 0.9% papain, 1% pectinase, and 4.0% cellulase by mass of polysaccharide to remove protein. After reacting for 2 h at their optimal enzyme activity temperature and pH range, respectively, the samples were adjusted to pH 7 with 6 mol/L NaOH. The enzyme was inactivated by increasing the temperature. After that, the polysaccharide samples were centrifuged, ethanol precipitated, and collected. The content of polysaccharides and protein was determined to calculate the polysaccharide retention rate and protein clearance rate.

| Sevage protein removal method
Eight equal parts of crude polysaccharide samples were taken, dissolved in distilled water, and named 1 ~ 8. Added the 1/5 volume times Sevage reagent (trichloromethane:n-butanol = 5: 1), vigorously shook for 20 min, then centrifuged (3500 r/min, 6 min, 4°C), and collected the upper layer. The other parts repeated this procedure 2 ~ 8 times. Then, the Lonicera japonica polysaccharides were precipitated with 4 volume absolute ethanol. After that, the polysaccharide retention rate and protein clearance rate were calculated.

| Enzymatic-Sevage protein removal method
The polysaccharide after removing protein by trypsin enzymolysis was taken, divided into eight parts, dissolved in distilled water, and named 1 ~ 8. The latter processes were similar to Sevage method and deproteinized eight parts for 1 ~ 8 times, respectively. Then the polysaccharide retention rate and protein clearance rate were calculated.

| TCA protein removal method
The Lonicera japonica crude polysaccharides were dissolved in distilled water, added the equal volume of 20% tricarboxylic acid (TCA) reagent, and intensely shook for 30 min. After waiting for 1 h at 4°C, the polysaccharide solution was centrifuged (3500 r/min, 6 min, 4°C), and adjusted to pH 7 with 6 mol/L NaOH, then precipitated and collected. The polysaccharide retention rate and protein clearance rate were calculated (Chen & Huang, 2018).

| TCA-n-butanol protein removal method
Taken two equal Lonicera japonica crude polysaccharides and dissolved in distilled water, named 1 and 2. Added twice volume (20% TCA:n-butanol = 1:10) reagent, shook for 15 min, and waited for 1 h at 4°C, collected the bottom layer and the two groups repeated this procedure 2 times. The polysaccharide was concentrated, precipitated, and dried and then calculated the polysaccharide retention rate and protein clearance rate.
The comprehensive score (Y) was regarded as the response. And the BBD is described in Table 1.

| Experimental design
After two days of adaptive feeding, 24 mice were randomly divided into four groups (n = 6), including normal group, model group, LNT group (treated with 30 mg/kg lentinan, LNT), and LJP group (treated with 800 mg/kg polysaccharide which deproteinized protein with the best method and the best condition). All groups received intragastric administration of 10 ml/kg each time for three days, while the normal group and model group were given the same volume of physiological saline. Then all groups were intraperitoneally injected with 50 mg/kg cyclophosphamide (CTX) once a day for three consecutive days, except the control group treated with the same volume of physiological saline, to build the CTX immunocompromised mice. All mice were allowed free access to food and water during the experiment. After successful modeling, all groups were continued to be administered for 10 days.

| Effect of LJP on mice organ index
After 12 h of the last treatment, the animals were weighed and blood was collected by removing the eyeballs, then the mice were sacrificed and collected the organs. The thymus and spleen were weighted and the indices were calculated as follows:

| Effect of LJP on IL-2 in serum and the level of serum hemolysin
On the seventh day of treatment, the mice began to be injected with 0.2 ml of 2% SRBC. After 4 days, the blood samples were collected from the eyes, then centrifuged, and the serum collected. Then the saline was used to multiple dilute the serum. Fifty microliters of 0.2% SRBC was added to the dilute mentioned above and incubated for 3 h at 37°C.
where 1, 2, 3, …, n, are multiple dilute indices and Ln is the degree of agglutination.
The IL-2 level in serum of mice was determined with ELISA kit according to the instructions provided by the manufacturer.

| Metabolite sample collection
After 12 h of the last treatment, the eyeballs were removed and blood was collected, then the mice were sacrificed. The blood was left at room temperature for 0.5 h and then centrifuged (12,000 r/ min, 15 min, 4°C) to isolate the serum and the plasma. The serum was collected, accurately transferred 150 μl, 450 μl methanol added, vortex-mixed for 60 s, and centrifuged (12,000 r/min, 15 min, 4°C).
Finally, the supernatant was injected into HPLC-TOF-MS (highperformance liquid chromatography time-of-flight mass spectrometry) for analysis.

| Chromatography conditions
Chromatographic analysis of serum samples was performed on the Halo-C 18 column (2.1 × 100 mm, 2.7 μm, Waters, USA). The column temperature was 35°C while the flow rate was 0.25 ml/min. The injection volume was 5 μl. The mobile phase used 0.1% aqueous formic acid (A) and acetonitrile (B). The gradient elution was as follows: 20%-85% B over 0-10 min, 85%-100% B over 11-20 min, and 100% B over 21-30 min. Mass spectrometry conditions were set as follows: the capillary voltage was 4000 V, the drying gas temperature was 350°C, and the gas flow rate was 8 L/min. The mass collection range was 50-1000 (Zhang et al., 2018). Organ index (mg ∕ g) = the organ weight (mg) ∕ body weight (g).

| Network pharmacology analysis
In order to further analyze the affection of biomarkers, MetScape for Cytoscape software, which can integrate biomolecular interaction networks with high-throughput expression data into a unified KEGGbased conceptual framework, was used to dig the upstream proteins of biomarkers. Meanwhile, the proteins related to immunomodulatory were collected based on the GeneCards database (https://www.genec ards.org/). These upstream proteins and pathological proteins were imported into STRING for protein-protein interactions analysis (PPI). The minimum required interaction score was set at 0.9 and hid disconnected nodes in the network to link their interactions. In this network, every node stood for a protein, and the edges stood for their relationship. analysis, the genes molecular functions information was determined.
Data visualization was carried out with a threshold (p < .05).

| Statistical analysis
All data obtained were summarized as mean ± standard deviation (SD) and statistical testing and comparisons were performed by using the one-way analysis of variance (ANOVA) method. Statistical analyses were done using SPSS 21.0. A value of p < .05 was considered as statistical significance.

| Deproteinization results' analysis of five methods
Lonicera japonica polysaccharides were deproteinized by the enzymatic method, Sevage method, enzymatic-Sevage method, TCA method, and TCA-n-butanol method, respectively. Considering the high polysaccharide retention rate and protein clearance rate, the trypsin method could be used for deproteinization as the best enzymatic method (Figure 2a). The results of the Sevage method are shown in Figure 2b, indicating that the appropriate deproteinization times were 6. The trypsin method combined with the Sevage method was also used to remove the protein (Figure 2c). To obtain high polysaccharide retention rate and protein clearance, 5 times of deproteinization was the appropriate time in the Trypsin-Sevage method. After using the TCA method, although the protein removal effect was ideal which reached 65.13%, the polysaccharide retention rate was only 39.32%, which may be due to more evident damage caused by TCA to Lonicera japonica polysaccharides. As presented in Figure 2d, after the protein was removed with TCA-n-butanol two times, the protein clearance rate increased, but the polysaccharide retention rate decreased. Considering the simplicity of the operation process, the frequency of protein removal with TCA-n-butanol was once.

| Comparison of the five methods for deproteinization
As shown in Figure 3, the best protein removal results in each method of Enzymatic, Sevage, Enzymatic-Sevage, TCA, and TCA-

| Statistical analysis and model fitting
Response surface methodology (RSM) is a collection of statistical and mathematical techniques useful for developing, improving, and optimizing processes (Liu, Ma, et al., 2015). A total of 17 designed experiments with three factors and three levels were conducted for optimizing the deproteinization conditions ( Table 2). The experiment data were analyzed by multiple regression analysis, and the secondorder polynomial equation was as follows: The analysis of variance (ANOVA) of the regression model is shown in

F I G U R E 3 Comparison of five methods' best results for deproteinization
were not a simple linear correlation. The order of the influence of single factors on the comprehensive score was polysaccharide solution: (TCA-n-butanol) (B) > TCA: n-butanol (A) > shaking time (C).

| Response surface analysis
To further explain the interaction between the independent variables and response values, three-dimensional (3D) response surface plots and two-dimensional (2D) contour plots are displayed in Figure 4. By analyzing these 3D response surface plots and their respective profiles, it was easy to understand the interaction between two independent variables and determine their optimal range (Liu, Ma, et al., 2015). As demonstrated in Figure 4, the polysaccharide solution: (TCA-n-butanol) had a significant effect on the comprehensive score. Meanwhile, the results showed that the polysaccharide solution: (TCA-n-butanol) and the shaking time have complex interaction effects.

| Model verification
By analyzing these data, the maximum comprehensive score of

| Immune organ index
The thymus and spleen are the main immune organs of the body, which affect the organism's immune function and the ability to resist disease. According to Figure 5a, the spleen and thymus indexes in the model group were much lower than those in the control group which illustrated that a weakened immune system was induced by CTX. After treatment with LJP, the spleen and thymus indexes were observably ameliorated, suggesting that LJP had a good immuneenhancing effect on the recovery of CTX damage to immune organs.

| IL-2 and serum hemolysin level
The immune response associated with levels of cytokines is a multistep process. IL-2 is an important cytokine that directly affects cellular immunity and humoral immunity. Under certain conditions, serum hemolysin would combine with SRBC and cause SRBC hemolysis (Dai et al., 2021). To further investigate the effect of LJP on humoral immunity, serum hemolysin level was also measured, and the results were presented as the antibody number. As shown in Figure 5b, the serum levels of IL-2 and antibody numbers in LJP group were significantly augmented compared with model group, suggesting that LJP could increase the secretion of IL-2 cytokine and reduce the severity of immunosuppression in mice.

| Multivariate data analysis
In this study, HPLC-TOF-MS method combined with SIMCA 13.0 was applied to analyze the serum of mice from normal, model, LNT, and LJP group. To further discover the differences among endogenous metabolites in each group, multivariate statistical analyses, including PLS-DA and OPLS-DA, were used (Hao et al., 2018).

| Metabolic pathway
To further explore the metabolic pathways of potential biomarkers related to LJP in the treatment of immunocompromised mice, MetaboAnalyst 5.0 was used to perform the pathway enrichment analysis against the potential biomarkers. As shown in Figure 8, the x-axis (pathway impact) stood for the importance of the metabolic pathway, and the y-axis (−log P) stood for the significance of the metabolic pathway enrichment analysis. The KEGG numbers of the 14 biomarkers were imported into MetaboAnalyst 5.0, and the impact value threshold of pathway topology analysis was set to 0.06, the values above this threshold were chosen as potential target pathways.

| Network pharmacology results
To further illustrate the potential mechanism of LJP on immunomodulatory effect, PPI was used to analyze the correlation between metabolic pathways and corresponding targets. As displayed in

| DISCUSS ION
As one of the oldest medicine systems in the world, TCM has the characteristics of multiple components and multiple targets and has shown great advantages in the treatment of various diseases . Some reports showed that as the important active ingredient of TCM, herbal polysaccharides have the characteristics of low toxicity and low side effects, and participate in many biological functions, such as antiviral, antitumor, antioxidant, hypolipidemic, immune regulation, and so on. Therefore, the separation, purification, and biological activity of TCM polysaccharides have become a research hotspot at home and abroad (Zeng et al., 2019). Lonicera japonica polysaccharides were used in the long course of medical practice. However, the polysaccharides extracted by traditional methods were usually impure and contained much protein, which affected the judgment of the activity of polysaccharides.
In this work, we investigated five methods to remove proteins: Enzymatic method, Sevage method, Enzymatic-Sevage, TCA method, and TCA-n-butanol method. We comprehensively explored the protein removal methods of Lonicera japonica polysaccharides from two aspects: the selection of deproteinized reagents and the number of deproteinized times. By comparing the best results of each method, it was determined that TCA-n-butanol method was the best method. In order to obtain better results of protein removal, we optimized the process by RSM. Based on the experimental data, the best protein removal condition was TCA:n-butanol = 1: 5.1, polysaccharide solution: (TCA-n-butanol) = 1:2.8, and shook for 33 min.
Thymus and spleen are the main central immune organs and the largest peripheral immune organs, respectively (Dai et al., 2021).
Both of them can directly reflect the level of immune function (Lv et al., 2019). The results showed that LJP could significantly promote the thymus and spleen indexes of mice. Interleukin-2 (IL-2) is F I G U R E 9 Protein-protein interactions analysis (PPI) network. The red and blue nodes represent the immune-related targets and potential biomarker-related targets, respectively. The red lines stand for the interaction between immune-related targets and potential biomarkers, while the black lines indicate the interaction between immune-related targets F I G U R E 1 0 The biological functions associated with the immunomodulatoryrelated targeted proteins and the potential biomarkers a kind of T-cell growth factor activity found in the supernatant of activated T cells in 1976 and is the main cytokine in the stimulation of T lymphocyte proliferation (Chen et al., 2006;Liao et al., 2011).
Serum hemolysin level is the main indicator reflecting nonspecific immune function, which reflects the proliferation and differentiation of B cells and their ability to secrete hemolysin after combining with complement . To investigate the effect of LJP on the immunosuppressed complement system, the level of IL-2 and serum hemolysin was measured. The results showed that the content of IL-2 and serum hemolysin was noticeably ameliorated after the LJP treatment.
Metabolomic is an emerging method that can reveal the specific changes in phenotypic physiological and biochemical states in biological systems. Hence, immunometabolism, notably amino acids, fatty acids, and glucose, is essentially needed for immune cells in homeostasis or pathological state as a new branch of metabolism.
The metabolites of some acids, such as tryptophan, are related to cell proliferation and growth processes. Amino acids play a crucial role as regulators and as main substrates in a variety of metabolic pathways (Babu et al., 2019). The upregulation of Glycolysis was a critical step in the activation of innate and adaptive immune cells, as it provides a means of increasing flux through the pentose phosphate pathway to synthesize macromolecules and generate the antimicrobial respiratory burst (Ganeshan & Chawla, 2014). Some reports showed that increased glycolysis could be considered a sign of metabolic changes in the rapid activation of most immune cells (O'Neill et al., 2016). Notably, existing reviews show that amino acid metabolism plays an important role in regulating the immune system. According to the metabolomics methods, pathways of differential metabolites in the LJP for the treatment of immunosuppressed mice were Citrate cycle, Glycolysis/Gluconeogenesis, and so on. Compared with the model group, LJP adjusted these main pathways to gradually restore the normal level of immunocompromised mice. At the same time, it was the first time to elucidate the mechanisms of action of LJP in immune regulation by the approach of network pharmacology. Nonetheless, further experiments are still needed to validate our findings.

| CON CLUS ION
In short, through optimization of conditions, we determined that the best method for removing protein of LJP was the TCA-n-butanol method, and the best condition was TCA:nbutanol = 1:5.1, polysaccharide solution: (TCA-n-butanol) = 1:2.8, and shook for 33 min. After the CTX-induced mice were treated with LJP, the thymus and spleen indexes were promoted, and the contents of IL-2 and hemolysin in mice serum were also restored.
Based on the serum metabolomics analysis, a total of 14 potential biomarkers about immune and LJP treatment have been identified in different groups. Taking these metabolites as the potential molecular targets, LJP could be inferred to partially recover the metabolism pathways of arginine biosynthesis, citrate cycle, and so on. Network pharmacology results suggested that LJP may protect immunosuppressed mice via the regulation of mainly ten biological processes including gluconeogenesis, tricarboxylic acid cycle, and so on. To sum up, our findings suggest that metabolomics method together with network pharmacology would be useful to explore the pathological mechanisms and clarify the mechanisms of the action of LJP for the treatment of immunomodulatory effects.

ACK N OWLED G M ENTS
This work was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest to declare.